Adenoviruses are small, non-enveloped viruses containing a linear double stranded DNA genome that were first discovered in 1953. The human adenoviruses are associated with a variety of diseases including upper respiratory infections, gastrointestinal illness, and conjunctivitis. Adenovirus infections are a significant clinical problem in transplant recipients, particularly pediatric patients. For many years, these viruses have also been outstanding model systems for the study of DNA replication, RNA synthesis, protein translation, oncogenic transformation, and apoptosis, and more recently interest in adenovirus has expanded due to its potential as a vector for vaccination and human gene transfer studies. Viral gene expression, genome replication, and viral assembly all take place in the nucleus of the infected cell. This project addresses assembly with a focus on the encapsidation of viral DNA. This process requires at least four viral elements: the packaging sequence, which is located near the left end of the genome and is made up of repeated elements called A repeats;the IVa2 protein, which binds to required sequence motifs in this region and is also required for capsid assembly;the L4 22 kDa protein, which forms a complex with the IVa2 protein on the DNA;and the 52/55 kDa protein, which binds both the DNA and the IVa2 protein. The goals of this proposal are to elucidate further the mechanism by which adenovirus encapsidates its DNA, and how this process is linked to capsid formation by the IVa2 protein. The mechanism by which the IVa2 protein facilitates capsid assembly and DNA packaging will be studied by examining the structure of complexes containing the IVa2 and L4 22 kDa proteins and by dissecting the various functions of the IVa2 protein. The role of the 52/55 kDa protein will also be uncovered through the analysis of mutant viruses. These studies will advance our basic understanding of how adenovirus packages its DNA and produces infectious virions. This knowledge will be applicable to the development of anti-viral drugs that block this process, as well as to the development of safer adenovirus vectors and recombinant vaccines.